Beta-globin gene cluster haplotypes in Venezuelan sickle cell patients from the State of Aragua

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Beta-globin gene cluster haplotypes in Venezuelan sickle cell patients from

the State of Aragua

Nancy Moreno

1

_{, José A. Martínez}

2

_{, Zorella Blanco}

3

_{, Leidys Osorio}

1

_{and Patrick Hackshaw}

4

1

_{Laboratorio de Biología Molecular, Universidad de Carabobo Núcleo Aragua.}

2

_{Departamento de Biología, Universidad Pedagógica Experimental Libertador Maracay, Estado Aragua.}

3

_{Hospital Dr. Domingo Luciani El Llanito.}

4

_{Servicio de Hematología del Hospital Central de Maracay, Estado Aragua.}

Abstract

Seven polymorphic sites in theβ-globin gene cluster were analyzed on a sample of 96 chromosomes of Venezuelan sickle cell patients from the State of Aragua. The Benin haplotype was predominant with a frequency of 0.479, followed by the Bantu haplotype (0.406); a minority of cases with other haplotypes was also identified: atypical Bantu A2 (0.042), Senegal (0.031), atypical Bantu A7 (0.021) and Saudi Arabia/Indian (0.021) haplotypes; however, the Cameroon haplotype was not identified in this study. Our results are in agreement with the historical records that establish Sudanese and Bantu origins for the African slaves brought into Venezuela.

Key words: haplotypes, Beta-globin gene cluster, sickle cell.

Received: March 5, 2002; accepted: March 25, 2002

Introduction

Sickle cell disease (SCD) exhibits a wide spectrum of clinical behavior: everything from a mild condition to an incapacitating illness; its hematological characteristics, as well as its clinical severity, are influenced by variations in fetal hemoglobin (HbF) levels (Falusi and Olatungi, 1994; Steinberg, 1996), the simultaneous presence of α-thalasse-mia (Figueiredo et al., 1996; Mukherjee et al., 1997) glu-cose-6- phosphate dehydrogenase deficiency (Bouanga et

al., 1998) and the β-globin cluster linked haplotypes

(Po-wars and Hiti, 1993; Po(Po-wars et al., 1994; Steinberg, 1996). The βS_{gene is in linkage disequilibrium with five main}

dif-ferent haplotypes of the β gene cluster, which were named according to the geographical areas where they are most prevalent: Benin, Cameroon, Central African Republic (CAR or Bantu), Senegal and Saudi Arabia/Indian (SAI) (Pagnier et al., 1984; Kulozik et al., 1986; Lapouméroulie

et al., 1992).

Sickle cell disease was introduced into the American continent mainly by the massive trade of African slaves which occurred between the sixteenth and nineteenth cen-turies, and is the most common hereditary disorder in the Americas. Sickle cell epidemiological studies carried out in

Venezuela demonstrated a variable frequency, ranging from zero in the Venezuelan Indians up to 5 percent in the Mestizo and Afro-American populations; in some regions such as the North Central Coastal Region the frequency has risen to as much as 12% (Arends, 1971; Arends et al., 1982). In order to provide the required information for fur-ther analysis of the heterogeneity of the hematological pa-rameters and clinical manifestations of SCD in Venezuela, the DNA β-globin gene cluster haplotypes were analyzed in 48 Venezuelan sickle cell patients from the State of Aragua. The results were compared with those found in other studies with Venezuelan patients (Arends et al., 2000), Guadeloupe Island (Kéclard et al., 1996) and Iberoamerican populations of Cuba (Muniz et al., 1995), Colombia (Cuellar-Ambrosi et al., 2000) and Brazil (Zago

et al., 1992; Figueiredo et al., 1994; Gonçalves et al., 1994;

Pante de Sousa et al., 1998, 1999).

Subjects and Methods

The patient sample was composed of 48 non-related HbS homozygotes, from the State of Aragua, which is in the North Central Coastal Region of Venezuela, at about 9°23’ to 10°33’ N. and 66°33’ to 67°53’ W. The population of this State is the result of an intense process of admixture among Caucasians, Amerindians, and Afro-Americans and its total population size, according to the 1990 census (OCEI, 1990) represents 6.2% of the Venezuelan

Genetics and Molecular Biology, 25, 1, 21-24 (2002)

Copyright by the Brazilian Society of Genetics. Printed in Brazil www.sbg.org.br

Send correspondence to Nancy Moreno. Apartado Postal 237, Maracay, Estado Aragua 2101, Venezuela. E-mail: nanmorja@ telcel. net.ve.

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population. These patients were attended at the Hematolog-ical Service at the Maracay Central Hospital in the State of Aragua. There were 23 males and 25 females, with a mean age of 21 ± 3.3 years.

Venous blood samples were collected using EDTA as an anticoagulant, and the hematological parameters were determined by standard laboratory procedures. The buffy coat was used for the preparation of nuclear DNA accord-ing to previously described methods (Kirby, 1992). Diag-nosis of SCD was based on the Bsu36I digestion of a PCR amplified 536 bp., verifying whether the β-globin DNA segment contained the βS_{mutation, as reported previously}

(Martínez et al., 1998). Haplotypes were determined by the analysis of the following polymorphic restriction sites in the β-globin gene cluster: 1) Hinc II 5’ of ε; 2) Xmn I 5’ of Gγ; 3) Hind III in the IVSII of Gγ; 4) Hind III in the IVSII of Aγ; 5) Hinc II in the Ψβ; 6) Hinc II 3’ of Ψβ; and 7) Hinf I 5’ β. Segments containing each of these sites were ampli-fied by PCR and subsequently digested with the appropri-ate enzyme. The oligonucleotide primers used for the analysis of sites 2 to 7 were described by Sutton et al. (1989) and site 1 was analyzed using a primer reported by Guerreiro et al. (1992).

Results

The β-globin cluster haplotypes identified in the Ven-ezuelan patients are presented in Table I. Benin is the most frequent with a frequency of 0.479, followed by Bantu (0.406), Senegal (0.031), and Saudi Arabia/Indian (0.021). The Bantu A2 and Bantu A7 atypical haplotypes described by Srinivas et al. (1988) were observed with a frequency of 0.042 and 0.021, respectively. No Cameroon haplotypes were identified in this study. Fourteen of the 48 SCD pa-tients were homozygous for the Benin haplotype (29.2%), 9 were Bantu homozygotes (18.7%) and all the others were

heterozygotes (52.1%). Table II shows the βS _typical

haplotype frequency distribution among the Venezuelan patients studied in this work, compared with those from several Iberoamerican populations and Caribbean Islands.

Discussion

The data reported in this study indicated that the Benin (47.9%) and Bantu (40.6%) haplotypes were the most prevalent, Senegal was present in 3.1% of the patients, a value similar to that found in the western region of Co-lombia (4.3%) and the Belém population of Brazil (3.4%). The frequencies of the two atypical haplotypes, Bantu A2 (4.2%) and Bantu A7 (2.1%), are some of the highest re-ported in American populations. This observation can be interpreted as resulting from of recombinatorial events in-volving the Bantu typical haplotype and the 5’ regions from various haplotypes present in normal individuals as de-scribed by Srinivas et al. (1988). In an admixed population such as the Venezuelan, there are more probabilities of ob-serving these events.

Fifty-two percent of the SCD patients studied pre-sented heterozygous haplotypes, as was expected due to the

22 Moreno et al.

Table I - β-globin gene cluster haplotypes identified in a sample of 96 chromosomes from Venezuelan sickle cell patients

Haplotype Number of chromosomes (n = 96) Frequency Benin 46 0.479 Bantu 39 0.406 Atypical Bantu A2 4 0.042 Senegal 3 0.031 Saudi Arabia/Indian 2 0.021 Atypical Bantu A7 2 0.021

Table II - Frequency distribution (%) of the typical βS_{haplotypes in patients from Iberoamerican populations and the Guadeloupe Island.}

Population N. of chromosomes Haplotypes

Bantu Benin Senegal Arab/Indian Cameroon

Venezuela (State Aragua)1 ₉₀ _43.3 _51.1 _3.3 _2.2 _0.0

Venezuela (All country)2 ₁₇₆ _32.4 _51.1 _14.2 _0.0 _2.3

Cuba3 ₁₉₈ _41.0 _51.0 _8.0 _0.0 _0.0

Colombia (Western Region)4 ₉₂ _55.5 _34.8 _4.3 _0.0 _5.4

Brazil (Afro-Brazilians from the Amazon Region)5 ₂₀ _60.0 _10.0 _30.0 _0.0 _0.0

Brazil (Belém, State of Pará)6,7 ₅₉ _66.1 _30.5 _3.4 _0.0 _0.0

Brazil (Salvador, State of Bahia)7 ₄₂ _55.0 _45.0 _0.0 _0.0 _0.0

Brazil (Ribeirão Preto, State of São Paulo)7,8 ₆₇ _73.0 _25.5 _1.5 _0.0 _0.0

Brazil (Campinas, São Paulo)9 ₁₄₂ _79.6 _18.4 _2.0 _0.0 _0.0

Guadeloupe10 ₂₅₂ _11.5 _77.0 _8.7 _0.0 _2.8

1_{Present study;}2_{Arends et al. (2002);}3_{Muniz et al. (1995);}4_{Cuellar-Ambrosi et al.(2000);}5_{Pante de Sousa et al. (1999);}6_{Pante de Sousa et al. (1998);} 7_{Figueiredo et al. (1994);}8_{Zago et al. (1992);}9_{Gonçalves et al. (1994).}10_{Kéclard et al. (1996).}

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admixture process which occurs in our population; in a study carried out on African SCD patients, the vast majority of haplotypes were found in homozygosity (Sow et al., 1995). Our results differed significantly from those re-ported by Arends et al. (2000) in another study performed on sickle cell Venezuelan patients from all over the country (χ2_{= 14.62; d.f. = 4; p < 0.01). This may be due to different}

patient ascertainment and/or a diverse historical origin of the African-derived patients from Aragua. On the other hand, our results are in agreement with the haplotype distri-bution found in patients from Cuba (χ2_{= 6.60; d.f. = 3;}

p > 0.05); while significant differences exist with the Co-lombian data (χ2_{= 11.23; d.f. = 4; p < 0.025). A highly}

sig-nificant difference was observed when the Venezuelan patients were compared with those from Guadeloupe (χ2 _{= 42.63 d.f. = 2; p < 0.001), and those from Brazil}

(χ2_{= 20.03; d.f.= 3; p < 0.001). It is probable that all these}

differences reflect the origin of the forced migration of Af-rican slaves to the AmeAf-rican continent, although, as was in-dicated for the two Venezuelan samples, patient ascertainment should also be considered.

African slavery was instituted in Venezuela to meet the growing labor demands of an emerging agricultural economy; historically it is reported that the African slaves brought to Venezuela were from the Sudan and Bantu re-gions (Acosta Saignes, 1984), although some slaves came to Venezuela from the others colonies, especially the Antil-les; in consequence, the predominance of the Benin and Bantu haplotypes is in accordance with these historical re-cords.

Acknowledgments

We are grateful to the blood donors for their collabo-ration with this investigation. We offer special thanks to Mrs Melanie Mackie and Lic. Jesús Ernesto Lisboa M. for their contribution in the manuscript revision. This research was supported by BID-CONICIT Grant BTS-067, CDCH Universidad de Carabobo Grant 90-007 and CIADANA.

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